Inhibition of endogenous phosphodiesterase 7 promotes oligodendrocyte precursor differentiation and survival.

During the development of the central nervous system (CNS), oligodendrocyte precursors (OPCs) are generated in specific sites within the neural tube and then migrate to colonize the entire CNS, where they differentiate into myelin-forming oligodendrocytes. Demyelinating diseases such as multiple sclerosis (MS) are characterized by the death of these cells. The CNS reacts to demyelination and by promoting spontaneous remyelination, an effect mediated by endogenous OPCs, cells that represent approximately 5-7 % of the cells in the adult brain. Numerous factors influence oligodendrogliogenesis and oligodendrocyte differentiation, including morphogens, growth factors, chemotropic molecules, extracellular matrix proteins, and intracellular cAMP levels. Here, we show that during development and in early adulthood, OPCs in the murine cerebral cortex contain phosphodiesterase-7 (PDE7) that metabolizes cAMP. We investigated the effects of different PDE7 inhibitors (the well-known BRL-50481 and two new ones, TC3.6 and VP1.15) on OPC proliferation, survival, and differentiation. While none of the PDE7 inhibitors analyzed altered OPC proliferation, TC3.6 and VP1.15 enhanced OPC survival and differentiation, processes in which ERK intracellular signaling played a key role. PDE7 expression was also observed in OPCs isolated from adult human brains and the differentiation of these OPCs into more mature oligodendroglial phenotypes was accelerated by treatment with both new PDE7 inhibitors. These findings reveal new roles for PDE7 in regulating OPC survival and differentiation during brain development and in adulthood, and they may further our understanding of myelination and facilitate the development of therapeutic remyelination strategies for the treatment of MS.

Cytoarchitectonic and neurochemical differentiation of the visual system in ethanol-induced cyclopic zebrafish larvae.

Embryonic exposure to ethanol leads to malformations such as cyclopia. Cyclopic embryos present fused eyes and lack of the ventral specification of the brain, with physiological and morphological defects in the visual system, which provides a useful model for teratology and neurotoxicity assessments. We analysed the differentiation of the visual areas in the ethanol-induced cyclopic animals. For this purpose we exposed zebrafish embryos to 1.5% ethanol from 4 hours post-fertilisation (hpf) to 24 hpf in order to get cyclopic embryos. We monitored cytoarchitecture and quantified both the proliferation rate and cell differentiation from 2 days post-fertilisation (dpf) onwards, focusing on the main components of the visual system (retina, optic nerve and optic tectum) of normal and cyclopic zebrafish embryos. The visual system of the zebrafish embryos is affected by exposure to ethanol; two optic nerves that fuse before leaving the eyes are present in cyclopic specimens but an optic chiasm is not evident. Cell differentiation is severely delayed throughout the visual system at 2 dpf. At 5 dpf, lamination in the cyclopic retina and optic tectum is completed, but they are filled with pyknotic nuclei demonstrating cell death. At this stage the proliferation rate and expression patterns are unaltered and glial and neuronal neurochemical differentiations are similar to untreated animals. We found that the alterations produced by exposure to ethanol are not only cell-selective, but also tissue-selective. Cyclopia is the most severe phenotype induced by ethanol, although cell differentiation and proliferation can reach normal patterns after a certain period of time, which points to a neural plasticity process. Zebrafish embryos may possess a compensation mechanism against the ethanol effect, which would account for their use for pharmacogenetic and chemical screenings in the analysis of new molecules that could improve visual problems.

Characterisation of neuronal and glial populations of the visual system during zebrafish lifespan.

During visual system morphogenesis, several cell populations arise at different time points correlating with the expression of specific molecular markers We have analysed the distribution pattern of three molecular markers (zn-1, calretinin and glial fibrillary acidic protein) which are involved in the development of zebrafish retina and optic tectum. zn-1 is a neural antigen expressed in the developing zebrafish central nervous system. Calretinin is the first calcium-binding protein expressed in the central nervous system of vertebrates and it is widely distributed in different neuronal populations of vertebrate retina, being a valuable marker for its early and late development. Glial fibrillary acidic protein (GFAP), which is an astroglial marker, is a useful tool for characterising the glial environment in which the optic axons develop. We describe the expression profile changes in these three markers throughout the zebrafish lifespan with special attention to ganglion cells and their projections. zn-1 is expressed in the first postmitotic ganglion cells of the retina. Calretinin is observed in the ganglion and amacrine cells of the retina in neurons of different tectal bands and in axons of retinofugal projections. GFAP is localised in the endfeet of Müller cells and in radial processes of the optic tectum after hatching. A transient expression of GFAP in the optic nerve, coinciding with the arrival of the first calretinin-immunoreactive optic axons, is observed. As axonal growth occurs in these regions of the zebrafish visual pathway (retina and optic tectum) throughout the lifespan, a relationship between GFAP expression and the correct arrangement of the first optic axons may exist. In conclusion we provide valuable neuroanatomical data about the best characterised sensorial pathway to be used in further studies such as teratology and toxicology.

Characterization of NADPH-diaphorase-positive glial cells of the tench optic nerve after axotomy.

NADPH-diaphorase (ND) positive cell types were characterized throughout the optic nerve of the tench in normal conditions and after optic nerve transection with survival periods of 1, 3, 7, 14, 30, 60, 120 and 180 days. Astrocytic markers (S100 and glutamine synthetase) and the microglial marker tomato lectin were employed. In the control prechiasmatic optic nerve two types (types I and II) of ND-positive glial cells appeared. All type I cells showed S100 immunoreactivity, whereas only a subpopulation of them were positive to glutamine synthetase. Type II cells only presented S100 immunoreactivity. In the control anterior optic tract, all ND-positive glial cells (type III) presented immunolabeling to S100 and glutamine synthetase. After transection, types I and II did not show any changes in the staining patterns for the glial markers when observed. Two new types of ND-positive glial cells (types IV and V) were observed after axotomy. All type IV cells were S100-immunopositive, and a subpopulation presented glutamine synthetase immunolabeling. Only a subpopulation of type V cells showed glutamine synthetase immunostaining. The presence of type IV or V cells in the lesioned optic nerve occurred simultaneously with significant decreases or absence of type I cells. These data suggest that type I and III cells are astrocytes and type II cells are oligodendrocytes. Types IV and V cells are the result of the activation of type I cells after optic nerve section. The polymorphism observed in ND-positive cells may reflect different cell functions during degenerative and regenerative processes.

Teratogenic effects of ethanol exposure on zebrafish visual system development.

Ethanol intake during pregnancy can produce a wide range of adverse effects on nervous system development including fetal alcohol syndrome (FAS). The most severe congenital malformation observed in newborns with FAS is cyclopia. In this study, we have exposed zebrafish embryos to different ethanol concentrations (2.4%, 1.5% or 1.0%) during eye morphogenesis in four zebrafish strains (AB, EK, GL and TL). In addition, we have studied the survival rate of the cyclopic animals to the end of larval development. The zebrafish strains GL and AB generated the higher percentage of cyclopic animals after exposure to 2.4% ethanol, while EK showed the higher percent cyclopic animals using 1.5% and 1.0% ethanol. The EK strain showed the higher percent survival during the larval period at all ethanol concentrations (2.4%, 1.5% and 1.0%). Moreover, we have investigated cytoarchitectural alterations in the main components of the visual pathway-retina and optic tectum-and ethanol treatment affects both the retina and the optic tectum. The lamination of neural retina is clearly delayed in treated larvae 3 days postfertilization and the thickness of the pigmented epithelium is considerably reduced. With regard to the optic tectum, treatment with ethanol alters the normal pattern of tectal lamination. The use of zebrafish EK strain is a suitable in vivo vertebrate model system for analyzing the teratogenic effect of ethanol during vertebrate visual system morphogenesis as it relates to both cyclopia and FAS.

Tyrosine hydroxylase immunoreactivity in the developing visual pathway of the zebrafish.

We analyzed the distribution of tyrosine hydroxylase immunoreactivity in the central nervous zones involved in the processing of visual information during zebrafish ontogeny, employing a segmental approach. In the retina, we observed immunolabeled cells in the inner nuclear layer after hatching. From the juvenile stages onwards, some of these cells presented two immunolabeled processes towards the inner and outer plexiform layers of the retina, which are identified as interplexiform cells. In the adult zebrafish retina, we have identified two cellular types displaying immunoreactivity for tyrosine hydroxylase: interplexiform and amacrine cells. In the optic tectum, derived from the mesencephalon, no immunolabeled neurons were observed in any of the stages analyzed. The periventricular gray zone and the superficial white zone display immunostained neuropile from the end of fry life onwards. At the 30-day postfertilization, the tyrosine hydroxylase immunoreactive neuropile in the optic tectum presents two bands located within the retinorecipient strata and deeper strata, respectively. All diencephalic regions, which receive direct retinal inputs, show immunolabeled cells in the preoptic area, in the pretectum, and in the ventral thalamus from embryonic stages onwards. During the fry development, the immunolabeled neurons can be observed in the periventricular pretectum from 15-days postfertilization and in both the ventrolateral thalamic nucleus and suprachiasmatic nucleus from 30-days postfertilization. The transient expression of tyrosine hydroxylase is observed in fibers of the optic tract during fry and juvenile development. The existence of immunolabeled neuropile in the zebrafish retinorecipient strata could be related to the turnover of retinotectal projections.

Comodulation of CXCR4 and CD26 in human lymphocytes.

We provide convergent and multiple evidence for a CD26/CXCR4 interaction. Thus, CD26 codistributes with CXCR4, and both coimmunoprecipitate from membranes of T (CD4(+)) and B (CD4(-)) cell lines. Upon induction with stromal cell-derived factor 1alpha (SDF-1alpha), CD26 is cointernalized with CXCR4. CXCR4-mediated down-regulation of CD26 is not induced by antagonists or human immunodeficiency virus (HIV)-1 gp120. SDF-1alpha-mediated down-regulation of CD26 is not blocked by pertussis toxin but does not occur in cells expressing mutant CXCR4 receptors unable to internalize. Codistribution and cointernalization also occurs in peripheral blood lymphocytes. Since CD26 is a cell surface endopeptidase that has the capacity to cleave SDF-1alpha, the CXCR4.CD26 complex is likely a functional unit in which CD26 may directly modulate SDF-1alpha-induced chemotaxis and antiviral capacity. CD26 anchors adenosine deaminase (ADA) to the lymphocyte cell surface, and this interaction is blocked by HIV-1 gp120. Here we demonstrate that gp120 interacts with CD26 and that gp120-mediated disruption of ADA/CD26 interaction is a consequence of a first interaction of gp120 with a domain different from the ADA binding site. SDF-1alpha and gp120 induce the appearance of pseudopodia in which CD26 and CXCR4 colocalize and in which ADA is not present. The physical association of CXCR4 and CD26, direct or part of a supramolecular structure, suggests a role on the function of the immune system and the pathophysiology of HIV infection.

A syndrome involving intrauterine growth retardation, microcephaly, cerebellar hypoplasia, B lymphocyte deficiency, and progressive pancytopenia.

We report a new complex syndrome involving profound failure to thrive with severe intrauterine growth retardation, cerebellar abnormalities, microcephaly, a complete lack of B lymphocyte development, and secondary, progressive marrow aplasia. B cell differentiation was found to be blocked at the pro-B cell stage. Although not strictly proven, a genetic origin is likely, according to similar cases reported in the literature. Three candidate genes, PAX5, encoding B cell-specific activator protein, a factor involved in B cell lineage commitment, stromal cell-derived factor 1, and CXCR4, encoding a chemokine and its receptor, respectively, were thought to be responsible for this disease, given the similarity between the phenotype of the corresponding knock-out mice and the clinical features of the patient. However, the genomic DNA sequences of these 3 genes were normal, and normal amounts of stromal cell-derived factor 1 and CXCR4 were present. These data strongly suggest that another molecule is involved in early B cell differentiation, hematopoiesis, and cerebellar development in humans.